Method for high resolution magnetic resonance analysis using magic angle technique

ABSTRACT

A method of performing a magnetic resonance analysis of a biological object that includes placing the object in a main magnetic field (that has a static field direction) and in a radio frequency field; rotating the object at a frequency of less than about 100 Hz around an axis positioned at an angle of about 54°44′ relative to the main magnetic static field direction; pulsing the radio frequency to provide a sequence that includes a phase-corrected magic angle turning pulse segment; and collecting data generated by the pulsed radio frequency. The object may be reoriented about the magic angle axis between three predetermined positions that are related to each other by 120°. The main magnetic field may be rotated mechanically or electronically. Methods for magnetic resonance imaging of the object are also described.

PRIORITY CLAIM

This application is a continuation-in-part of U.S. application Ser. No.09/803,381, filed Mar. 9, 2001, incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support underContract DE-AC0676RLO1830 awarded by the U.S. Department of Energy. TheUnited States Government has certain rights in the invention.

FIELD

The present disclosure relates to magnetic resonance (MR) analysis,particularly to magnetic resonance spectroscopy (MRS) and imaging (MRI)of biological objects.

BACKGROUND

Magnetic resonance is a phenomenon exhibited by a select group of atomicnuclei and is based upon the existence of nuclear magnetic moments inthese nuclei (termed “gyromagnetic” nuclei). When a gyromagnetic nucleusis placed in a strong, uniform and steady magnetic field (a so-called“external field” and referred to herein as a “static” magnetic field),it precesses at a natural frequency known as a Larmor frequency. TheLarmor frequency is characteristic of each nuclear type and is dependenton the applied field strength in the location of the nucleus. Typicalgyromagnetic nuclei include ¹H (protons), ¹³C, ¹⁹F and ¹³P. Theprecession frequencies of the nuclei can be observed by monitoring thetransverse magnetization that results after a strong RF pulse applied ator near their Larmor frequencies. It is common practice to convert themeasured signal to a frequency spectrum by means of Fouriertransformation.

More specifically, when a bulk sample containing nuclear magneticresonance (NMR) active nuclei is placed within a magnetic field, thenuclear spins distribute themselves amongst the nuclear magnetic energylevels in accordance with Boltzmann's statistics. This results in apopulation imbalance between the energy levels and a net nuclearmagnetization. It is this net nuclear magnetization that is studied byNMR techniques.

At equilibrium, the net nuclear magnetization is aligned parallel to theexternal magnetic field and is static. A second magnetic fieldperpendicular to the first and rotating at, or near, the Larmorfrequency can be applied to induce a coherent motion of the net nuclearmagnetization. Since, at conventional field strengths, the Larmorfrequency is in the megahertz frequency range, this second field iscalled a “radio frequency” or RF field.

In particular, a short (microsecond) pulse of RF radiation is applied tothe sample in the static magnetic field; this pulse is equivalent toirradiating at a range of frequencies. The free induction decay (FID) inresponse to the RF pulse is measured as a function of time. The responseof the sample to the pulse depends upon the RF energy absorption of thesample over a range of frequencies applied (for example, 500 MHz±2500Hz). Often the pulse is applied many times and the results are averagedto improve the signal-to-noise ratio.

The coherent motion of the nuclear magnetization about the RF field iscalled a “nutation.” In order to deal conveniently with this nutation, areference frame is used which rotates about the z-axis at the Larmorfrequency. In this “rotating frame” part of the RF field, which isrotating in the stationary “laboratory” reference frame in the samedirection as the magnetization, is static. Consequently, the effect ofthe RF field is to rotate the nuclear magnetization direction at anangle with respect to the main static field direction. By convention, anRF field pulse of sufficient length to rotate the nuclear magnetizationthrough an angle of 90° or π/2 radians is called a “π/2 pulse.”

A π/2 pulse applied with a frequency near the nuclear resonancefrequency will rotate the spin magnetization from an original directionalong the main static magnetic field direction into a planeperpendicular to the main magnetic field direction. The component of thenet magnetization that is transverse to the main magnetic fieldprecesses about the main magnetic field at the Larmor frequency. Thisprecession can be detected with a receiver coil that is resonant at theprecession frequency and located such that the precessing magnetizationinduces a voltage across the coil. Frequently, the “transmitter coil”employed for generating the RF field to the sample and the “receivercoil” employed for detecting the magnetization are one and the samecoil.

In addition to precessing at the Larmor frequency, in the absence of theapplied RF field, the nuclear magnetization also undergoes tworelaxation processes: (1) the precessions of various individual nuclearspins which generate the net nuclear magnetization become dephased withrespect to each other so that the magnetization within the transverseplane loses phase coherence (so-called “spin-spin relaxation”) with anassociated relaxation time, T₂, and (2) the individual nuclear spinsreturn to their equilibrium population of the nuclear magnetic energylevels (so-called “spin-lattice relaxation”) with an associatedrelaxation time, T₁. The spin-spin relaxation is caused by the presenceof small local magnetic fields, arising from the electrons, magneticnuclei, and other magnetic dipoles surrounding a particular nucleus.These fields cause slight variations in the resonance frequency of theindividual nuclei, which results in a broadening of the NMR resonanceline. Often this broadening is caused by two types of local fields: astatic component, which gives rise to a so-called inhomogeneousbroadening, and local fields which are fluctuating in time as a resultof molecular motions and interactions between magnetic nuclei. Thelatter phenomenon results in a so-called homogeneous broadening.

Magnetic resonance imaging and magnetic resonance spectroscopy are usedextensively in biological research and medicine, both for in vitroinvestigations of cells and tissues and for in vivo measurements onanimals and humans. Both methods are non-invasive and non-destructiveand are used for a large variety of applications, including thedetection and diagnosis of lesions and diseases, and the evaluation oftherapy response. One particularly useful MRS technique is ¹H nuclearmagnetic resonance (NMR) spectroscopy. ¹H NMR spectroscopy has been usedextensively to study metabolic changes in diseased cells and tissues andthe effects of therapy. The resonance lines corresponding to several keymobile compounds have been observed, and their spectral intensities havebeen linked to the tumor phenotype, tumorigenesis, tumor size, increasedproliferation of cells, cell apoptosis, and necrosis.

However, a serious problem associated with these applications is therelatively large widths of the MR resonance lines that are observedusing conventional MRI and MRS. This reduces the MRI and MRSsensitivity, and, for MRS, can result in severely overlapping spectrallines, which seriously hampers the analysis of the spectrum. It has beenestablished that in biological materials the line widths are mainlycaused by inhomogeneous broadening. In intact cells and tissues, thepossible mechanisms that broaden the lines inhomogeneously includeresidual chemical shift anisotropy interaction and local magnetic fieldgradients arising from variations in the bulk magnetic susceptibility atthe various compartment boundaries present in the cells and tissues. Itis believed in the art that the bulk magnetic susceptibility variationsare the main mechanisms responsible for the broadening. Using cellextracts can eliminate this broadening, but this procedure cannot beapplied in live subjects, it is time consuming and may introducespectral artifacts.

It is well known that the susceptibility broadening and otherinhomogeneous broadening mechanisms can be eliminated by magic anglespinning (MAS), where the sample is rotated about an axis with an angleof 54° 44′ (or cos⁻¹ (3^(−½))) with respect to the static magnetic fielddirection. A problem with MAS is that when the value of the spinningrate is small compared to the width of the broadening, the resonant peaksplits into a group of spinning sidebands (SSBS) separated by thespinning rate. If the value of the spinning rate is less than theisotropic spectral width, the analysis of the spectra becomesconsiderably difficult due to the overlapping of the SSBs associatedwith the different resonant peaks. This problem can be avoided byincreasing the spinning rate to eliminate the SSBs in the spectralregion of interest. Indeed it has been shown that fast MAS, where asample is rotated at a speed of several kHz, produces a significantnarrowing of the MR lines in cells and tissues (see Weybright et al.,Gradient, High-Resolution, Magic Angle Spinning ¹ H Nuclear MagneticResonance Spectroscopy of Intact Cells, Magnetic Resonance in Medicine1998; 39: 337-345; and Cheng et al., Quantitative Neuropathology by HighResolution Magic Angle Spinning Proton Magnetic Resonance Spectroscopy,Proc. Natl. Acad. Sci. USA 1997; 94: 6408-6413). However, the largecentrifugal force associated with such high spinning rates destroys thetissue structure and even part of the cells (see Weybright et al.).Consequently, MAS at a high spinning speed is not suitable, for example,to map the metabolite distribution in intact biological tissues or tostudy live cells, and is impossible to use on live subjects.

A possible way to overcome the problems associated with fast MAS is touse slow sample spinning. Many methods have been developed in solidstate NMR to eliminate the spinning sidebands or to separate them fromthe isotropic spectrum so that a sideband free isotropic chemical shiftspectrum is obtained. One approach is the so-called magic angle turning(MAT) techniques, and sideband free isotropic chemical shift spectrahave been obtained in solids at spinning rates as low as 30 Hz (Hu etal., Magic Angle Turning and Hopping, in Encyclopedia of MagneticResonance D. M. Grant, and R. K. Harris, Eds. New York: John Wiley &Sons: 1996, 2914-2921).

MAT is a two dimensional (2D) NMR technique that was developed todetermine the chemical shift tensors of rare spins such as ¹³C and ¹⁵Nin solids. There are basically two types of MAT experiments. The firsttype (MAT-1) is based on the Magic Angle Hopping (MAH) experimentpioneered by Bax et al., Correlation of Isotropic Shifts and ChemicalShift Anisotropies by Two-Dimensional Fourier-Transform Magic-AngleHopping NMR Spectroscopy, J. Magn. Reson. 1983; 52: 147. The secondclass (MAT-2) involves the use of five radio frequency π pulses during aconstant evolution time period (e.g., one rotor period). MAT-2techniques include the five π replicated magic angle turning (FIREMAT)(Hu et al., An Isotropic Chemical Shft-Chemical Shift Anisotropy MagicAngle Slow-Spinning 2D NMR Experiment, J. Magn. Reson. 1993; A 105:82-87; and Alderman et al., A sensitive, high resolution magic angleturning experiment for measuring chemical shft tensor principal values,Molecular Physics 1998; 95(6): 1113-1126) and the 2D-phase-alteredspinning sidebands (PASS) techniques (Antzutkin et al., Two-DimensionalSideband Separation in Magic-Angle-Spinning NMR,. J. Magn. Reson 1995; A115: 7-19). All of these experiments are 2D isotropic-anisotropicchemical shift correlation experiments yielding a high resolutionisotropic chemical shift dimension and a chemical shift anisotropydimension. Although MAT has been applied in solid state NMR (see Hu etal., Magic Angle Turning and Hopping; Gan et al., High-ResolutionChemical Shift and Chemical Shift Anisotropy Correlation in Solids UsingSlow Magic Angle Spinning, J. Am. Chem. Soc. 1992; 114: 8307-8309; Hu etal., Magic-Angle-Turning Experiments for Measuring Chemical-Shift-TensorPrincipal Values in Powdered Solids, J. Magn. Reson. 1995: A 113:210-222; Hu et al., An Isotropic Chemical Shift-Chemical ShiftAnisotropy Magic Angle Slow-Spinning 2D NMR Experiment; Alderman et al.,A Sensitive, High Resolution Magic Angle Turning Experiment forMeasuring Chemical Shift Tensor Principal Values; and Antzutkin et al.,Two-Dimensional Sideband Separation in Magic-Angle-Spinning NMR), itspotential for biological research has not been explored.

One of the reasons that MAT for biological objects, as opposed to solidobjects, has not been investigated is the belief that the diffusion ofthe molecules containing the nuclei of interest in the internal staticlocal magnetic fields results in a time-dependent field as experiencedby the nuclei. This effect worsens if the spinning frequency is reduced,resulting in imperfect suppression of the SSB's. In other words, it wasexpected that MAT techniques could not be employed in biologicalmaterials because the Brownian motions, which cause metabolites todiffuse throughout the cells, would make it impossible to remove thesusceptibility broadening with slow MAS. Indeed, it was shown that in astandard fast MAS experiment of water embedded in glass beads thespectral lines become broad even at spinning speeds of several hundredHz (see Leu et al, Amplitude Modulation and Relaxation Due to Diffusionin NMR Experiments With a Rotating Sample, Chem Phys Lett 2000;332:344-350), and that a sideband-suppression technique called totalsuppression of sidebands (TOSS) was ineffective for suppressing SSB'sarising from water embedded in glass beads when the spinning speed waslowered to 1 kHz (see Liu et al, Manipulation of Phase and AmplitudeModulation of Spin magnetization in Magic Angle Spinning NMR in thePresence of Molecular Diffusion, J. Chem. Phys. 2001: 114: 5729-5734).

Another approach for increasing the sensitivity and resolution of NMRspectroscopy involves rotating the magnetic field rather than thesample. According to this approach the sample remains stationary. Forexample, Bradbury et al., Nuclear Magnetic Resonance in a RotatingMagnetic Field, Phys. Letters 1968; 26A: 405-406, disclose rotating thestatic magnetic field by superposing a static field and two sinusoidalfields in phase quadrature in the plane perpendicular to the staticfield and with amplitudes that are a factor 2 larger than that of thestatic component. However, this approach was never considered anyfurther.

Thus, a need exists for a method for obtaining high resolution magneticresonance analysis of biological objects. In particular, there is a needfor a magnetic resonance analysis technique that does not damage tissueor cell structure in biological objects and avoids the problemsassociated with SSBs at slow object spinning rates.

SUMMARY

Described herein are methods for magnetic resonance analysis of anobject by combining slow magic angle spinning techniques with certainradio frequency pulse sequences. This combination provides for the firsttime a method for obtaining high resolution spectra of a biologicalobject that (a) does not damage tissue or cellular structure in thebiological object and (b) substantially eliminates spinning sidebandpeaks in the spectra associated with slow magic angle spinning. Contraryto the conventional expectation that the diffusion of the moleculescontaining the nuclei of interest in the internal static local magneticfields would be problematic for slow spinning, the inventors havesurprisingly discovered that the presently disclosed methods provide NMRspectra with a resolution comparable to or better than the spectralresolution obtained with conventional fast MAS, and that aresubstantially free of spinning sidebands peaks at low rotationfrequencies.

In particular, according to a first embodiment there is provided amethod of performing a magnetic resonance analysis of a biologicalobject that includes placing the biological object in a main magneticfield and in a radio frequency field, the main magnetic field having astatic field direction; rotating the biological object at a rotationalfrequency of less than about 100 Hz around an axis positioned at anangle of about 54°44′ relative to the main magnetic static fielddirection; pulsing the radio frequency to provide a sequence thatincludes a magic angle turning pulse segment; and collecting datagenerated by the pulsed radio frequency.

According to a second embodiment there is provided a method ofperforming a magnetic resonance analysis of a biological object thatincludes placing the biological object in a main magnetic field and in aradio frequency field, the main magnetic field having a static fielddirection; rotating the biological object at a rotational frequency ofless than about 100 Hz around an axis positioned at an angle of about54°44′ relative to the main magnetic static field direction; pulsing theradio frequency to provide a sequence capable of producing a spectrumthat is substantially free of spinning sideband peaks; and collectingdata generated by the pulsed radio frequency.

According to a third embodiment there is provided a method of performinga magnetic resonance analysis of a biological object that includessubjecting the biological object to a static magnetic field and a pulsedradio frequency field, the main magnetic field having a static fielddirection; rotating the biological object at a rotational frequency ofless than about 100 Hz around an axis positioned at an angle of about54°44′ relative to the main magnetic static field direction; controllingthe pulsed radio frequency to provide a sequence of pulses of radiofrequency radiation capable of producing a spectrum that issubstantially free of spinning sideband peaks; and generating a magneticresonance analysis of the response by nuclei in the biological object tothe pulsed radio frequency sequence.

According to a fourth embodiment there is provided a method ofperforming a magnetic resonance analysis of a biological object thatincludes placing the biological object in a main magnetic field and in aradio frequency field, the main magnetic field having a static fielddirection; positioning the object along a magic axis located at an angleof about 54°44′ relative to the main magnetic static field direction;reorienting the object about the magic angle axis between threepredetermined positions, the three predetermined positions being relatedto each other by 120°; pulsing the radio frequency to provide a sequencecapable of producing a spectrum that is substantially free ofanisotropic broadening (e.g., from magnetic susceptibility); andcollecting data generated by the pulsed radio frequency.

According to a fifth embodiment there is provided a method of performinga magnetic resonance analysis of a biological object that includesproviding a main magnetic field that includes a first component having astatic field direction and an amplitude and a second and a thirdcomponent, each second and third component having a sinusoidal field ina plane perpendicular to the static field direction of the firstcomponent and with an amplitude that is 2^(½) times the amplitude of thestatic field of the first component, wherein the second and thirdcomponents produce a magnetic field that rotates in a planeperpendicular to the static field direction at a frequency of less thanabout 100 Hz resulting in an overall field that is rotating around anaxis located at an angle of about 54°44′ relative to the static fielddirection of the first component; placing the biological object in themain magnetic field and in a radio frequency field; pulsing the radiofrequency to provide a pulse sequence capable of producing a spectrumthat is substantially free of spinning sideband peaks; and collectingdata generated by the pulsed radio frequency.

According to a sixth embodiment there is provided a method of performinga magnetic resonance analysis of a biological object that includesplacing the biological object in a main magnetic field and in a radiofrequency field, the main magnetic field having a static fielddirection; mechanically rotating a magnet around an axis at an angle ofabout 54°44′ relative to the main magnetic static field direction at arotational frequency of less than about 100 Hz; pulsing the radiofrequency to provide a sequence capable of producing a spectrum that issubstantially free of spinning sideband peaks; and collecting datagenerated by the pulsed radio frequency.

According to a seventh embodiment there is provided a method ofperforming a magnetic resonance analysis of a biological object thatincludes placing the biological object in a main magnetic field and in aradio frequency field, the main magnetic field having a static fielddirection; rotating the biological object at a rotational frequency ofless than about 50 Hz around an axis positioned at a magic angle ofabout 54°44′ relative to the main magnetic static field direction;rotating the main magnetic field at a rotational frequency of less thanabout 50 Hz around the magic angle axis such that the main magneticfield and the biological object rotate simultaneously in the oppositerotational direction; pulsing the radio frequency to provide a sequencecapable of producing a spectrum that is substantially free of spinningsideband peaks; and collecting data generated by the pulsed radiofrequency.

According to an eighth embodiment there is provided a method forperforming a magnetic resonance imaging of a biological object thatincludes subjecting the biological object to a main magnetic field thathas a static field direction, a pulsed radio frequency field, and atleast one pulsed magnetic field gradient. The biological object isrotated at a rotational frequency of less than about 100 Hz around anaxis positioned at an angle of about 54°44′ relative to the mainmagnetic static field direction. The pulsed radio frequency iscontrolled to provide a pulse sequence that includes a magic angleturning pulse segment. The pulsed radio frequency and pulsed magneticfield gradient are also pulsed to generate spatially-selective nuclearmagnetic resonance data. A magnetic resonance analysis of the responseby nuclei in the biological object to the pulsed radio frequencysequence is generated.

It has been found that one particularly useful variant of the methodsdisclosed herein involves utilizing a pulse sequence that includes a2D-phase-altered spinning sidebands (2D-PASS) pulse segment. Anotherparticularly useful pulse segment is a phase-corrected magic angleturning (PHORMAT) pulse segment.

For in vitro investigations of small objects, the methods that include a2D-PASS segment are especially useful for increasing the NMR sensitivityin a MRI experiment, and for increasing the sensitivity and resolutionof NMR spectra of ¹H and other NMR-sensitive nuclei in MRS experimentsin cells and intact excised tissues and organs. For in vivoinvestigations of larger biological objects, the methods that include aPHORMAT segment are especially useful for increasing the resolution ofNMR spectra of ¹H and other NMR-sensitive nuclei in MRS experiments inlive animals and humans. The slower rotating of the sample minimizes, ifnot substantially eliminates tissue and cellular damage. The presentlydisclosed methods have several important advantages over fast MAS: (I)larger rotors and, henceforth, larger samples can be used, whichincreases the NMR sensitivity (especially important when the method isapplied for less NMR-sensitive nuclei than protons); (II) the structuralintegrity of the biological sample undergoes minimal or no changes underslow spinning (i.e., artifacts in the spectra induced by the fastspinning, which are a result of the sample deformation during thespinning, are avoided); and (III) besides the isotropic spectrum, theanisotropy patterns of the individual water and metabolite lines can bedetermined (allowing one to obtain information regarding the immediatesurroundings of the various compounds).

BRIEF DESCRIPTION OF THE DRAWINGS

Certain embodiments will be described in more detail with reference tothe following drawings:

FIG. 1 is a perspective view of rotating a biological object at themagic angle relative to the main static magnetic field;

FIG. 2 represents one embodiment of a 2D-PASS RF pulse sequence inaccordance with the presently disclosed methods;

FIGS. 3A and 3B show ¹H spectra obtained by analyzing an excised intactmouse brain using conventional NMR techniques and a stationary sample;

FIGS. 3C and 3D show ¹H spectra obtained by analyzing an excised intactmouse using slow MAS but with RF pulse sequencing that did not include awater suppression segment and a MAT segment;

FIG. 4 shows a stacked plot of a ¹H 2D-PASS spectra obtained byanalyzing an excised intact mouse brain using one embodiment of thepresently disclosed methods;

FIGS. 5A, 5B and 5C show ¹H 2D-PASS spectra obtained by analyzing anexcised intact mouse brain using one embodiment of the presentlydisclosed methods;

FIGS. 5D and 5E show ¹H spectra obtained by analyzing an excised intactmouse brain using prior art fast MAS;

FIGS. 6A, 6B and 6C show proton spectra of a stationary sample obtainedby analyzing an excised intact mouse brain with a RF pulse sequence thatdid not include a water suppression segment;

FIGS. 7A-7H show proton spectra of different excised intact mouse organsand tissues obtained with 2D-PASS;

FIG. 8 represents a further embodiment of a RF pulse sequence (PHORMAT)in accordance with the presently disclosed methods;

FIGS. 9A and 9B show ¹H PHORMAT spectra obtained by analyzing excisedrat liver tissue using one embodiment of the presently disclosedmethods;

FIGS. 10A, 10B and 10C show ¹H spectra obtained by analyzing excised ratliver tissue using embodiments of the presently disclosed methods;

FIG. 10D shows ¹H spectra obtained by analyzing excised rat liver tissueusing fast MAS;

FIGS. 11A, 11B and 11C show ¹H spectra obtained by analyzing excised ratliver tissue using embodiments of the presently disclosed methods;

FIG. 11D shows ¹H spectra obtained by analyzing excised rat liver tissueusing fast MAS;

FIG. 12 is a schematic representation of a RF coil configuration forelectronically rotating the magnetic field;

FIG. 13 is a schematic representation of a device that holds thebiological object stationary and mechanically rotates the magneticfield;

FIGS. 14A-14E schematically represent embodiments of pulse sequencesthat combine MRI sequences with 2D-PASS sequences; and

FIGS. 15A and 15B schematically represent embodiments of pulse sequencesthat combine MRI sequences and PHORMAT sequences.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

For ease of understanding, the following terms used herein are describedbelow in more detail:

“Object” means a three-dimensional object such as an intact animal, ananimal organ, a solid object such as an archaeological artifact, aspectrographic sample such as a tissue or cellular slice, a liquidnon-biological material such as an organic compound or a solid materialsuch as a metallic powder.

“Fluid object” means an object that includes a substantial amount offluid (such as greater than about 60 weight %.), as opposed to a solidobject. A typical example of a fluid object is an intact human or ahuman organ that typically includes at least about 80 weight % water.

“Biological object” means any object, usually a fluid object, thatincludes cellular matter. Exemplary biological objects include cellsystems, excised tissues and intact organs, live animals, and humanpatients.

“Main magnet” or “main magnetic field” denotes the magnet that generatesthe static magnetic field (typically referred to as B₀ or H₀) as knownin the art. The main magnetic field is distinguished from the RFmagnetic field used to induce excitation of the atomic nuclei or the RFmagnetic gradient field used in magnetic resonance. Of course, MRS andMRI tools that could be used with the described method include a mainmagnet capable of producing the static and homogeneous main magneticfield. Such magnets are well known and typically are superconductingmagnets.

The above definitions are provided solely to aid the reader, and shouldnot be construed to have a scope less than that understood by a personof ordinary skill in the art or as limiting the scope of the appendedclaims.

Magic angle spinning is based on exposing the object to a partiallytime-dependent external magnetic field rather than the static magneticfield B₀ currently used. Specifically, the magnetic field consists of astatic component with amplitude B₀/{square root over (3)}, and acomponent of amplitude B₀⅔, rotating in a plane perpendicular to thestatic field component.

In accordance with the first, second and third above-identifiedembodiments the slow magic angle spinning involves spinning or rotatingthe object at a frequency of less than about 100 Hz, preferably lessthan about 10 Hz, and more preferably less than about 3 Hz. An exampleof a range of possible rotating frequencies is about 1 to about 100 Hz,preferably about 1 to about 5 Hz. In contrast, standard fast magic anglespinning employs frequencies on the order of at least one kHz.

In accordance with the fourth above-identified embodiment the object is“hopped” over angles of 120° around the magic axis rather thancontinuously rotated. The time to complete one full rotation correspondsto the rotation times when spinning continuously (e.g., as in the first,second and third embodiments) at a frequency of less than about 100 Hz,preferably less than about 10 Hz, and more preferably less than about 3Hz.

In accordance with the fifth above-identified embodiment both the magnetand the biological object remain stationary, and part of the magneticfield is rendered electronically time dependent such that the overallmagnetic field is rotated around the magic axis with respect to thedirection of the overall magnetic field at a frequency of less thanabout 100 Hz, preferably less than about 10 Hz, and more preferably lessthan about 3 Hz. An example of a range of possible rotating frequenciesis about 1 to about 100 Hz, preferably about 1 to about 5 Hz.

In accordance with the sixth above-identified embodiment the biologicalobject remains stationary, and the magnet is physically rotated aroundthe magic angle with respect to the direction of the main magnetic fieldat a frequency of less than about 100 Hz, preferably less than about 10Hz, and more preferably less than about 3 Hz. An example of a range ofpossible rotating frequencies is about 1 to about 100 Hz, preferablyabout 1 to about 5 Hz.

In accordance with the seventh above-identified embodiment both thebiological object and the magnetic field are rotated in oppositedirections, each at a frequency of less than about 50 Hz, preferablyless than about 3 Hz, and more preferably less than about 2 Hz. Anexample of a range of possible rotating frequencies is about 0.5 toabout 50 Hz, preferably about 0.5 to about 2 Hz.

An example of a configuration for rotating the object while the mainstatic magnetic field is stationary is shown in FIG. 1. A biologicalobject 1 is placed in a sample holder 2 that is rotatable about an axis3 in a direction X placed in a static magnetic field generated by a mainmagnet (not shown) in a MRS or MRI tool. Axis 3 is located at an angleof 54°44′ relative to the direction of the static magnetic field B₀. MRS(e.g., NMR) and MRI apparatus capable of rotating an object or samplefor MAS are well known (see e.g., U.S. Pat. No. 4,511,841). Commerciallyavailable NMR tools that have rotors for spinning a sample include thoseprobes available from Varian/Chemagnetics, Inc. (Ft. Collins, Colo.) orBruker Instruments, Inc. (Billerica, Mass.).

An example of a magnet configuration for electronically rotating themagnetic field while the biological object is held stationary per theabove-described fifth embodiment is shown in FIG. 12. A complementarypair of first RF coils 21 are arranged to generate an alternatingmagnetic field B_(x) in the x-direction, given by

B _(x)=⅔·B ₀·sin(ω_(r) t).

A complementary pair of second RF coils 22 are arranged to generate astatic magnetic field B_(y) in the y-direction, given by

B_(y) =B ₀/3.

A complementary pair of third RF coils 23 are arranged to generate analternating magnetic field

B _(z)=⅔·B ₀·cos(ω_(r) t).

DC and AC currents passing through each set of coils 21, 22, 23 producethree mutually orthogonal magnetic field components. A biological object20 is placed in the center of the coil system. As a result, the overallmagnetic field is given by B₀, which rotates around an axis making themagic angle of 54°44′ relative to the static component B_(y).

An example of a configuration for physically rotating the magnet whilethe biological object is held stationary per the above-described sixthembodiment is shown in FIG. 13. A magnet bore 10 defines a void 11 forreceiving a biological object (e.g., a human) 12 and a longitudinal axis13. The longitudinal axis 13 of the magnet bore 10 is aligned at themagic angle of 54°44′ relative to the direction of the main magneticfield B₀. The main magnetic field B₀ is generated by the magnet bore 10.The magnet bore 10 may be rotated mechanically around the longitudinalaxis 13 as shown by the directional arrow in FIG. 13.

The above-described seventh embodiment involves rotating both thebiological object and the main magnetic field in respectively oppositerotational directions. For example, the device depicted in FIG. 12 orFIG. 13 could be modified so that the biological object also rotates.Such rotation allows for the rotational frequency of both the biologicalobject and the main magnetic field to decrease by a factor of two.

The RF pulse sequence employed in the presently disclosed methods may beany sequence or series of sequences capable of producing ahigh-resolution spectrum in a slow MAS approach that is substantiallyfree of spinning sidebands. The RF pulse sequences can be repeatedduring every rotor period (i.e., one 360° rotation of the object)throughout the duration of the scanning. A typical characteristic ofthese RF pulse sequences may be isotropic-anisotropic chemical shiftcorrelation pulse sequences. Exemplary RF pulse sequences include MATsequences. These RF pulse sequences preferably can be appliedsynchronously with the spinning of the object. A combination of RF pulsesequences that each have a different function may be used.

One example of a MAT technique that could be used in the disclosedmethods involves continuously rotating the biological object andapplying five RF π pulses during a constant evolution time period (e.g.,one rotor period). A π pulse rotates the magnetization over 180°.

An illustrative five RF π it pulses technique is the five π itreplicated magic angle turning (FIREMAT) as described, for example, inHu et al., An Isotropic Chemical Shift-Chemical Shift Anisotropy MagicAngle Slow-Spinning 2D NMR Experiment, J. Magn. Reson. 1993; A 105:82-87; and Alderman et al., A Sensitive, High Resolution Magic AngleTurning Experiment for Measuring Chemical Shift Tensor Principal Values,Molecular Physics 1998; 95(6): 1113-1126. Another illustrative five RFit pulses technique is the 2D-phase-altered spinning sidebands (PASS)technique as described, for example, in Antzutkin et al.,Two-Dimensional Sideband Separation in Magic-Angle-Spinning NMR,. J.Magn. Reson 1995; A115: 7-19). One variant of a 2D-PASS segment is shownin FIG. 2. All of these experiments are 2D isotropic-anisotropicchemical shift correlation experiments yielding a high resolutionisotropic chemical shift dimension and a chemical shift anisotropydimension.

A further example of a particularly useful MAT technique is known asphase-corrected magic angle turning (PHORMAT) as described, for example,in Hu et al., Magic-Angle-Turning Experiments for MeasuringChemical-Shift-Tensor Principal Values in Powdered Solids, J. Magn.Reson. 1995: A 113: 210-222 and Hu et al., Magic Angle Turning andHopping, in Encyclopedia of Magnetic Resonance, D. M. Grant and R. K.Harris, Eds. New York: John Wiley & Sons: 1996, 2914-2921. Similar to2D-PASS, PHORMAT involves continuously rotating the object with RFpulses spaced at one-third of the rotor period in order to obtain anisotropic shift evolution. According to the PHORMAT technique, echopulses are incorporated into the pulse sequence in such a way that themagnetization refocuses exactly, despite the modulation of the chemicalshift by the rotation of the sample. In particular, two pulse sequencesare employed that are derived from a combination ofmixed-amplitude-phase-modulated and triple-echo sequences. Thesesequences have the effect of converting phase modulation to amplitudemodulation, creating the equivalent of positive and negative evolutiontimes by placing the 180° echo pulses either before or after the threephase-accumulating periods.

With either PASS or PHORMAT the isotropic peak can be separated from theSSB's at all spinning speeds, and the linewidth is substantiallynarrowed even at a spinning speed as low as about 1 Hz.

In PASS the magnetization is constantly present in the transverse plane,and the first signal is observed after one rotor period. The amplitudeof the signal may be reduced as a result of the decay of themagnetization during this period, which is governed by the spin-spinrelaxation time T₂. Therefore, signal attenuation may occur when thespinning rate is comparable to or less than (T₂)⁻¹.

In PHORMAT the magnetization is stored longitudinally along the mainfield direction with a maximum duration of ⅔ times the rotor period.Consequently, the spinning frequency has to be large compared with thespin-lattice relaxation rate (T₁)⁻¹ of the spins in order to avoidsignal attenuation. Lower MAS frequencies can be used with PHORMATrelative to PASS since (T₁)⁻¹ is usually an order of magnitude smallerthan (T₂)⁻¹ in biological objects For example, PASS is particularlyuseful for spinning frequencies of greater than about 10 Hz, especiallyat least about 20 Hz, and PHORMAT is particularly useful for spinningfrequencies of less than about 10 Hz (e.g., about 1 Hz to 50 Hz). Afurther distinction between PHORMAT and PASS is that the measuring timeof a PASS analysis can take only a few minutes, but the measuring timeof a PHORMAT analysis can take up to one hour or more. Yet anotherdistinction between PHORMAT and PASS is that in a PHORMAT experiment theNMR sensitivity is reduced by at least an inherent factor 4 relative toPASS.

Another RF pulse sequence that is useful in the presently detailedmethods is a water suppression segment that suppresses residual SSBscaused by water in a biological object. The water suppression pulsesequence may be used for analysis of metabolite spectra. Without watersuppression these metabolite spectra would be polluted by artificiallines arising from residual SSBs of the water. Of course, watersuppression is not used when it is desired to investigate the water peakor signal of a biological object. An exemplary water suppression segmentis a DANTE pulse sequence as described, for example, in Morris et al.,Selective Excitation in Fourier Transform Nuclear Magnetic Resonance, J.Magn. Reson., 1978; 29:433-462. Another possible water suppressionsequence is the known combination of a shaped pulse segment and a pulsedfield gradient segment as described in Chen et al., Biochemical AnalysisUsing High-Resolution Magic Angle Spinning NMR SpectroscopyDistinguishes Lipoma-like Well-differentiated Liposarcoma from NormalFat, J. Am. Chem Soc. 2001; 123:9200-9201.

According to an example of the presently disclosed methods, the RF pulsesequence includes a DANTE pulse sequence segment followed by a 2D-PASSsequence segment as shown in FIG. 2. In this instance, thecross-polarization component described in Antzutkin et al.,Two-Dimensional Sideband Separation in Magic-Angle-Spinning NMR, wasreplaced by a π/2 pulse to rotate the magnetization in a planeperpendicular to that of B₀. In a 2D-PASS spectrum, the sidebandstypically are separated by the order n of the SSB. The center-bandspectrum, where n=0, displays a SSB-free spectrum, whereas the otherspectra show the SSB spectra in increasing order. By shifting theside-band spectra by n times the spinning frequency and adding them alltogether, an isotropic spectrum may be obtained. As was explained inAntzutkin et al., Two-Dimensional Sideband Separation inMagic-Angle-Spinning NMR, an intrinsic T₂ weighting of one rotor periodcan be introduced in 2D-PASS. In order to compensate for pulseimperfections and RF field inhomogeneity, a phase cycling sequence maybe applied. A preferred phase cycle consists of about 96 steps, and issubstantially the same as originally developed for the FIREMATexperiment (see Alderman et al., A Sensitive, High Resolution MagicAngle Turning Experiment for Measuring Chemical Shift Tensor PrincipalValues). It has been found that fewer phase steps could be used in theinvention without major spectral distortions. The timings tm₁-tm₆between the six pulses may be determined by the so-called PASS-16sequence given in Antzutkin et al., Two-Dimensional Sideband Separationin Magic-Angle-Spinning NMR. Sixteen different combinations of delaytimes tm₁-tm₆ were used (to be called evolution increments hereafter),which makes it possible to separate the centerband and 15 sidebandspectra without spectral aliasing. The width of the π pulse variestypically on the order of tens of microseconds to a millisecond,depending on the type and the amount of tissue loaded into the rotor. InFIG. 2 time point “T” denotes the end of a rotor period, time point “0”denotes the start of a rotor period and “acq” denotes acquisition of theNMR signal. The timings were counted from the middle of the it pulses.Two extra data points were acquired at the beginning of the acquisitiondimension to account for the dead time effect associated with probe ringdown and receiver recovery, which were not included in the Fouriertransformation. Fourier transformation using only 16 points along theevolution dimension was applied.

According to another example of the presently disclosed methods, the RFpulse sequence includes a modified PHORMAT sequence as depicted in FIG.8. The 90° pulses labeled (I), (II) and (III) are synchronized to ⅓ ofthe rotor cycle by marking the rotor evenly with three precision marksthat are mutually 120° apart. An optical detector is used to generatetransistor-to-transistor logic (TTL) pulses associated with thesemarkers, which serve as trigger pulses for the RF pulse sequences. Thepresence of the markers means that the spinning speed has to be stableonly for a short period of time, i.e., during one rotor period. Theposition of the rotor with respect to the external field may differ by0°, 120° or 240° at the beginning of each evolution increment. In thecase of an anisotropic object, such rotor positioning may createdistortions in the spectra as well as a loss in sensitivity. This issuecould be overcome by putting a single extra marker on the rotor andusing a second optical generator to generate a TTL pulse associated withthis marker to trigger the beginning of a PHORMAT sequence.

A 90° pulse (I) is substituted for the cross-polarization component torotate the magnetization in a plane perpendicular to that of B₀. A DANTEpulse sequence segment was employed immediately before the last pulse(III) to suppress the water signal. The DANTE pulse sequence wasinserted by switching the carrier frequency to the center of the waterpeak prior to the start of the DANTE sequence and then switching thisfrequency back to its original value at the end of the DANTE segment.The first two 90° pulses (I) and (II) are delayed by the time τ of theDANTE segment applied before the third readout pulse (III). This delayis instituted to separate the three read pulses (I, II, and III) byexactly ⅓ of the rotor period.

In FIG. 8 the 90° pulses are denoted in black and the 180° pulses aredenoted in gray. The phase cycling for the initial 90° pulse labeled by“a” is (−y, +y) while the phase cycles for the rest of the pulses (p₁,p₂, b₁, b₂ and b₃) are the same as those described in Hu et al.,Magic-Angle-Turning Experiments for Measuring Chemical-Shift-TensorPrincipal Values in Powdered Solids, J. Magn. Reson. 1995: A 113:210-222. The parameter A denotes half of the echo time. The use of 180°pulses, placed before (+) of after (−) the three phase accumulationperiods significantly improves the base plane of the 2D spectra andproduces a spinning sideband-free isotropic spectrum directly as aprojection onto the evolution axis without shearing. The time variablest₁ and t₂ correspond to the evolution and acquisition dimension,respectively. The bottom trace is the TTL signal generated by theoptical sensor of the MAS probe.

Alternative methods for water suppression employing pulsed fieldgradients can be used that significantly shorten the τ values (see Chenet al., Biochemical Analysis Using High-Resolution Magic Angle SpinningNMR Spectroscopy Distinguishes Lipoma-like Well-differentiatedLiposarcoma from Normal Fat, J. Am. Chem Soc. 2001; 123:9200-9201) sothat a higher spinning rate can be achieved with a PHORMAT sequence.

The fourth embodiment mentioned above in the Summary utilizes atechnique generally known in the art as magic angle hopping (MAH). Inparticular, the object is quickly reoriented (i.e., “hopped”) about themagic angle axis between three predetermined positions being related toeach other by 120°. One methodology for accomplishing this reorientationis to hop or rotate the biological object three times (e.g., 0-120degrees, 120-240 degrees and 240-0 degrees, or 0-120 degrees, 120-240degrees and 240-360 degrees) about an axis at the magic angle. The RFfrequency may be pulsed to produce a high-resolution spectrum that issubstantially free of line broadening caused by the bulk magneticsusceptibility and the residual chemical shift interaction. IllustrativeMAH techniques and the accompanying RF pulse sequences are described,for example, in Bax et al., Correlation of Isotropic Shifts and ChemicalShift Anisotropies by Two-Dimensional Fourier-Transform Magic-AngleHopping NMR Spectroscopy, J. Magn. Reson. 1983; 52: 147; Hu et al.,Improving the Magic Angle Hopping Experiment, Solid State NMR, 2,235-243 (1993); and Hu et al., Magic Angle Turning and Hopping, inEncyclopedia of Magnetic Resonance D. M. Grant, and R. K. Harris, Eds.New York: John Wiley & Sons: 1996, 2914-2921. A water suppression RFpulse sequence as described above could also be used in connection withMAH techniques.

The fifth embodiment mentioned above in the Summary utilizes a rotatingmagnetic field that is a superposition of a static field and twoorthogonal sinusoidal fields in phase quadrature in the planeperpendicular to the static field and with amplitudes that are a factor2^(½) larger than that of the static component. In particular, three RFcoil configurations are used to produce magnetic fields in threemutually perpendicular directions. Applying a stationary current to oneof the coils and quadrature sinusoidal AC currents to the other twocoils electronically generates a rotating magnetic field. A magneticfield component is generated that is rotated in a plane perpendicular tothe direction the static field component and with an amplitude that is2^(½) times the amplitude of the static field component. The resultingoverall magnetic field rotates at a frequency of about 1 to about 100Hz, preferably about 1 to about 10 Hz, with the angle between the staticfield direction and the direction of the overall rotating magnetic fieldbeing about 54°44′. In other words, a magnetic field is created thatrotates relative to a stationary object. Furthermore, by making theamplitude of the sinusoidal fields 2^(½) times larger than thestationary or static field, the resulting magnetic field rotates at therequired magic angle.

The eighth embodiment described above in the Summary relates to MRI(including localized MRS) methods that may be enhanced by utilizing theslow magic angle turning techniques disclosed herein. In this instance,the biological object is also subjected to pulsed magnetic fields thatcan produce gradients in the main magnetic field in the X, Y and Zdirections. This method provides the ability of obtaining nuclearmagnetic resonance data concerning a specific predetermined region orspace of the biological object rather than the whole object.

One example of combining MAT with MRI involves applying a MAT sequenceas described above to a biological object rotating around the magic axisto obtain a NMR spectrum by generating magnetic field gradients rotatingsynchronously with the object and preceding the MAT sequence with volumeselective RF and gradient pulses such as point resolved surfacespectroscopy (PRESS) (see Bryant et al., Spatial Localization Techniquesfor Human MRS, Biomedical Magnetic Resonance Imaging and Spectroscopy(Young, ed. Wiley, New York, pp. 785-791 (2000)). U.S. Pat. No.4,301,410 describes a system and process for generating magnetic fieldgradients that rotate synchronously with an object. Specificillustrations of combining MRI sequences with slow MAT sequences aredepicted in FIGS. 14A-14E and 15. FIGS. 14A-14E show various examples ofimaging pulse sequence combined with a PASS sequence. FIGS. 15A and 15Bshow pulse sequences that include a PHORMAT sequence.

FIG. 14A shows a 2D imaging pulse sequence combined with a PASSsequence. The π/2 pulse is a sinc selective pulse applied in thepresence of the gradient Gz. Gradients Gx, Gy and Gz are rotatingsynchronously with the sample rotation so that in the sample frame thegradients are static. The gradient coils themselves are not required torotate since the rotating gradients may be obtained electronically withac currents through the coils, similar to the way a rotating B₀ field isproduced electronically. Gz is the slice selection gradient along therotor axis. Gx is the readout gradient along the rotor x axis. Gy is thephase encoding gradient along the rotor y axis. For water suppression, aCHESS sequence (Haase et al., ¹ HNMR Chemical Shift Selective (CHESS)Imaging, Phys. Med. Biol. 1985; 30:341-344; Dreher et al., Changes inApparent Diffusion Coefficients of Metabolites in Rat Brain After MiddleCerebral Artery Occlusion Measured by Proton Magnetic ResonanceSpectroscopy, Magn. Reson. Med. 2001; 45:383-389 can be used to replacethe DANTE segment.

FIG. 14B depicts a 2D chemical shift imaging pulse sequence utilizing aPASS sequence. The only difference with the 2D-MRI-PASS sequence shownin FIG. 14A is that the readout gradient Gx is replaced by aphase-encoding gradient in the same direction.

FIG. 14C shows an example of a 3D imaging pulse sequence combined withthe PASS method. Gradients Gx, Gy and Gz are rotating synchronously withsample rotation so that in the sample frame the gradients are static asdescribed above in connection with FIG. 14A. Gz is the phase encodinggradient along the rotating axis, Gy is the phase encoding gradientalong the rotor y axis, and Gx is the readout gradient along the rotor xaxis. For water suppression, a CHESS sequence can be used to replace theDANTE segment.

FIG. 14D illustrates a 3D chemical shift imaging pulse sequenceutilizing a PASS sequence. The only difference with the 2D-MRI-PASSsequence shown in FIG. 14C is that the readout gradient Gx is replacedby a phase-encoding gradient in the same direction.

FIG. 14E shows an example of volume-selected localized magneticresonance spectroscopy MRS using PASS. Gx, Gy and Gz are rotatinggradients that are rotating synchronously with the sample rotation asdescribed above in connection with FIG. 14A. After the DANTE sequence, atailored excitation sequence (Ernst et al., Principles of NuclearMagnetic Resonance in One and Two Dimensions, Oxford University PressInc., New York, 1997, p. 557) is applied such that the RF spectrum isessentially white except for a dip. Simultaneously, a x-gradient isapplied. As a result, all volume elements are saturated except for aslice perpendicular to the x-axis. Then the tailored excitation isrepeated in the presence of a y-gradient. As a result, only a tubeperpendicular to the y-axis is not saturated. Finally, a selective sinc90° pulse is applied in the presence of a z-gradient and a volume istherefore excited. For water suppression, a CHESS sequence can be usedto replace the DANTE segment.

FIG. 15A shows a localized PHORMAT sequence using rotating gradients.Sequence (a) is the basic PHORMAT sequence that includes the trigger(b), where the pulses located at (I, II and III) positions are sincselective pulses in the presence of gradients (c) (analogous to thestimulated echo acquisition (STEAM) sequence, see J Frahm et al., J.Magn. Res. 72, 502 (1987)). The black pulses are non-selective 90°pulses and the shaded pulses are non-selective 180° pulses. Thegradients labeled with ‘*’ are spoil gradients used to destroy anymagnetization that is left in the transverse plane after the blackpulses. The (c) pulsed gradients are rotating synchronously with theobject. Hence Gx, Gy, and Gz are static in a reference frame rotatingsynchronously with the object as described above in connection with FIG.14A. For water suppression, a CHESS sequence can be used to replace theDANTE segment.

FIG. 15B depicts a localized PHORMAT sequence using a static gradient.Sequence (a) is the basic PHORMAT sequence that includes the trigger(b), where the pulses located at (I, II and III) positions are sincselective pulses applied at the presence of gradient pulses (c). Theblack pulses are non-selective 90° pulses and the shaded pulses arenon-selective 180° pulses. Only the static z-gradient needs to be usedsince the three sinc pulses are located 120° around the circle of samplerotation provided that the rotation axis (MA) is along the magic angleas illustrated in (d). The gradients labeled with ‘*’ are spoilgradients used to destroy any magnetization that is left in thetransverse plane after the black pulses. For water suppression, a CHESSsequence can be used to replace the DANTE segment.

The RF radiation utilized in the pulse sequence of the presentlydisclosed methods can be generated by RF coils in a MR apparatus asknown in the art. The RF pulse sequencing may be generated by techniquesknown in the art. For example, most modem NMR and MRI spectrometers havepulse programmers and amplifiers that are capable of producing thesequences.

The data for generating a spectrographic analysis based on the disclosedmethod can be collected by the same coil used for generating the RFradiation, or by a separate receiver coil. A graphical representation ofthe collected data may be generated by techniques known in the art suchas, for example, software programs available on most modem NMR and MRIspectrometers.

The specific examples described below are for illustrative purposes andshould not be considered as limiting the scope of the appended claims.

EXAMPLE 1 Sample Preparation

Fresh tissues were excised from four mice, which were genetic mutants of129/SvJ mice cross-bred with C57BI/6J. The mice used carry hereditaryhemochromatosis, a single genetic mutation that results in excess irondeposition in tissues if excess iron is provided in the mouse ration.Tissues analyzed were considered normal, as the ration provided thesemice had normal levels of iron. Forty-five days old male and female miceweighing 20 grams were sacrificed by cervical dislocation, tissues wererapidly removed and either immediately analyzed (brain and liver) orstored from two to four hours at 2° C. before analysis (kidney, heartand gluteus muscle). The tissues were inserted into a 7.5-mm outerdiameter, 5-mm inner diameter pencil rotor commercially available fromChemagnetics Inc., and were held between two TEFLON plugs in the centerof the rotor. All experiments were performed at about 25° C.

Results

¹H NMR experiments were performed on a Chemagnetics 300 MHz Infinityspectrometer, with a proton Larmor frequency of 299.982 MHz. A standardcross-polarization (CP)/MAS probe with a 7.5-mm pencil type spinnersystem and an air-flow restriction in the driver channel was used. Inthis way it was possible to regulate the spinning rate with an accuracyof about ±2 Hz over a spinning range from 43 to 125 Hz. Followingconventional practice, the ¹H spectra are expressed in “ppm” which meanspart per million of the spectrometer operating frequency.

FIGS. 3A and 3B show the Bloch decay 1D spectrum, obtained without watersuppression on a static sample of a freshly excised mouse brain inaccordance with conventional NMR techniques. The experiments wereperformed within 15 minutes after the tissue was excised. The spectrawere acquired following the excitation of a RF pulse with a tip angle ofabout 10 degrees. The delay between the end of the pulse and the startof data acquisition was 20 μs. FIG. 3A shows the static spectrum andFIG. 3B shows the same spectrum with a magnification factor of 32. Awater line and some barely visible metabolite peaks can bediscriminated. The spectral resolution is poor.

FIG. 3C shows the 1D spectrum of an excised mouse brain tissueundergoing MAS at a frequency of 43 Hz but with a RF pulse sequence thatdid not include a water suppression segment and a MAT segment. FIG. 3Dshows the corresponding 32-fold magnification. The line width (FWHM) ofthe center band is approximately 13 Hz, which is significantly less thanthat (105 Hz) of the stationary spectrum. However, the sideband familiesfrom the metabolites are superimposed with each other and are alsosuperimposed with SSBs from the water resonance (FIG. 3D), making theassignment of the spectrum impossible.

FIG. 4 shows the stacked plot of the ¹H 2D-PASS spectrum of the samebrain tissue as in FIG. 3 acquired at a sample spinning rate of 43 Hz.In this case water suppression was used. This was achieved by applyingthe DANTE pulse sequence at the center band of the water spectrum. Inthis way, both the signal arising from the center band and the SSBs aresaturated. The parameter n denotes the nth sideband, n=0 corresponds tothe center band. The spectrum was acquired 24 minutes after the brainwas excised. Sixteen evolution increments were used, each with 96 phaseincrements, resulting in a total of 1,536 acquisitions. The recycledelay time was 2s, resulting in an experimental time of about 52minutes. The ¹H π/2 pulse width was 9 microseconds. The DANTE sequencecontained 4000 pulses spaced by 100 μmicroseconds, each of which was 1microsecond. In FIG. 4 ω₂ denotes the acquisition dimension, ω₁ denotesthe evolution dimension and ω_(r) denotes the angular spinningfrequency.

FIG. 5A shows the (water-suppressed) proton spectrum, obtained byprojecting the 2D-PASS data into the normal acquisition (ω₂) dimension.This spectrum reflects the result of a standard 1D experiment applied at43 Hz. Due to the overlapping of the SSB families from differentmetabolites, even with water suppression such a 1D spectrum is difficultto interpret. FIGS. 5B and 5C display the n=0 center-band spectrum andthe isotropic projection, respectively. Despite the relatively short T₂weighting time of one rotor period used in the 2D-PASS experiment (˜23.3msec in this case), an impressive spectral resolution is observed,indicating that the line broadening observed in the brain can be removedefficiently using the 2D-PASS experiment. The relative intensities ofthe various lines in the isotropic projection spectrum (FIG. 5C) areslightly different from those in the center-band spectrum (FIG. 5B),which is a result of differences in the anisotropy patterns of thevarious lines. Also, the spectral resolution in the isotropic spectrumis somewhat less than that of the center band. This is due to the slightinstability of the spinning rate, which has almost no effect on thecenter-band spectrum but broadens the lines in the side-band spectra,increasing with the order of the sideband. FIG. 5D shows the brainspectrum, obtained from a standard 1D fast MAS experiment at a spinningrate of 4.3 kHz. It follows that despite the much larger spinning ratethe spectral resolution is actually less than that of the center-bandspectrum obtained from 2D-PASS. This is, in part, due to the intrinsic23.3-msec T₂ weighting employed in the latter experiment. This is shownin FIG. 5E, where the same spectrum is shown after using a 23 msec T₂weighting, obtained by applying a π pulse train. Even in this case thewidth of the lines are about 8 Hz broader than those in the center-bandspectrum of 2D-PASS, causing an apparent relative intensity drop for thetwo narrowest resonance lines at 2.0 and 3.0 ppm, which are fromN-acetylaspartate and creatine, respectively. This extra broadening isprobably caused by an increased B₀inhomogeneity along the spinning axisinduced by the 4.3 kHz spinning, which is not averaged out by thespinning. The fast spinning pushes the sample against the rotor wall andcreates a hole in the middle, resulting in increased bulk susceptibilitygradients at the boundary of the sample and the hole.

FIGS. 6A, 6B and 6C illustrate the effects of such rapid samplespinning. In this figure the static water line is shown before spinning(FIG. 6A), after spinning at 43 Hz (FIG. 6B), and after spinning at 4.3kHz (FIG. 6C). It follows that the slow spinning hardly affects the lineshape, but that the sample deformation due to the fast spinning causessevere line broadening. We found that repacking the rotor after the fastspinning produced a similar spectrum as shown in FIG. 6A, proving thatthe sample deformation is the cause of this broadening. Hence, in orderto avoid this effect in a fast spinning experiment it is necessary touse densely-packed samples in a spherical sample holder. By using slowsample spinning as in the presently described methods, this problem isavoided.

FIGS. 7A and 7B show the spectra on a mouse heart using 2D-PASS andwater suppression at 80 Hz according to the presently disclosed method(7A) and using 1D-MAS and water suppression at 4.4 kHz (7B). FIGS. 7Cand 7D show the spectra on a mouse liver using 2D-PASS and watersuppression at 100 Hz according to the presently disclosed method (7C)and using 1D-MAS and water suppression at 3.3 kHz (7D). FIGS. 7E and 7Fshow the spectra on a mouse gluteus muscle using 2D-PASS and watersuppression at 125 Hz according to the presently disclosed method (7E)and using 1D-MAS and water suppression at 4.2 kHz (7F). FIGS. 7G and 7Hshow the spectra on a mouse kidney using 2D-PASS and water suppressionat 100 Hz according to the presently disclosed method (7G) and using1D-MAS and water suppression at 5.7 kHz (7H). It is clear from FIGS.7A-7F that for the heart, liver, and gluteus muscle both slow MAS andfast MAS methods provide spectra with very similar resolutions andintensities. In the kidney (FIGS. 7G and 7H) the lines obtained withfast spinning are somewhat broader than those obtained with slow MAS,which may be caused again by extra susceptibility gradients imposed bythe spinning.

These results demonstrate that the presently disclosed slow spinningmethods produce spectral resolutions that are similar, and in some caseseven better, compared to spectral resolutions obtained with fast MAS.

EXAMPLE 2 Sample Preparation

The experiments described below were performed on excised rat livertissues, obtained from Fisher 344 male rats. The liver was chosenbecause the proton lines obtained on static samples are found to be sobroad that resolution of the various metabolites is difficult orimpossible. Prior to removing the livers from the animals, the rats weresacrificed by CO₂ asphyxiation. Before loading the sample into the NMRrotor, about 200 mg of liver was cut into small pieces (approximately 2mm in size), and randomly selected aliquots were inserted into the rotorto provide a more or less isotropic sample.

Two different sample preparations were used. In the first case theexcised liver was loaded into the NMR rotor immediately after thecutting. Hence the results obtained in this sample may be an indicationof what can be expected in in vivo experiments. In the second case theexcised liver was prepared in the same way as described in Bollard etal., High-Resolution ¹ H and ¹ H-¹³ C Magic Angle Spinning NMRSpectroscopy of Rat Liver, Magn. Reson. Med. 2000; 44:201-207, i.e., theliver was perfused with saline to remove residual blood and was thensnap-frozen using liquid nitrogen and stored at −80° C. until required.Also, prior to the actual experiments the frozen sample was kept in therotor for about 19 hours. It was found that the sample degradationassociated with this procedure causes a significant increase in thespectral resolution, albeit at the cost of serious changes in thevarious line intensities. Hence this sample provides a more sensitiveway of comparing the spectral resolution obtainable with the variousexperiments than the first one. The liver pieces were loaded between twoTeflon plugs within a 7.5-mm OD, 5-mm ID Chemagnetics pencil rotor. Theparts were slowly pushed into the rotor to avoid large air bubbles inthe sample region in the rotor. All experiments were performed at roomtemperature, i.e. 25° C.

Results

¹H NMR experiments were performed on a Chemagnetics 300 MHz Infinityspectrometer, with a proton Larmor frequency of 299.982 MHz. A standardChemagnetics CPIMAS probe with a 7.5-mm pencil type spinner system wasused. In order to be able to spin at low frequencies, the rotor wasequipped with a flat drive tip (i.e. it did not contain grooves, whichare normally used to drive the rotor) and an airflow restriction wasused in the driver channel. The spinning rate was controlled using acommercial Chemagnetics MAS speed controller under the automated controlmode. Spinning rates as high as 5 kHz could be reached after removingthe airflow restriction in the driver channel and by replacing the fiatdrive tip with a standard tip. Following conventional practice, the ¹Hspectra are expressed in “ppm” which means part per million of thespectrometer operating frequency.

A modified PHORMAT sequence as depicted in FIG. 8 was applied to the ratlivers. The echo time (Δ) and the recycle delay times were 50 μs and 1s, respectively. The free-induction decays in the acquisition dimension(t₂) contained 300 complex points and were transformed to spectra with aspectral width of 8 kHz. The 2D data were collected using 100 t₁ steps,incremented 700 μs, corresponding to a maximum evolution time of 70 msand an evolution spectral width of 1.282 kHz. 2D data sets were acquiredwith the (+) and the (−) PHORMAT pulse sequences using a total of 64scans at each t₁ value, resulting in a total measuring time of about 3.0hours. Hyper-complex 2D data sets were constructed according to theprocedure detailed in Hu et al., Magic-Angle-Turning Experiments forMeasuring Chemical-Shift-Tensor Principal Values in Powdered Solids, J.Magn. Reson. 1995: A 113: 210-222 using a macro driven program developedon the Chemagnetics Infinity Spectrometer. The pulse width was 9.5 μs.The DANTE sequence consisted of 2000 RF pulses spaced by 100 μs and witha pulse width of 0.8 μs for each pulse, resulting in a cumulative flipangle of 15,200 degrees and a τ value of approximately 202 ms.

FIGS. 9A and 9B show ¹H PHORMAT spectra of the fresh liver sample,obtained at a spinning rate of 1 Hz. FIG. 9A displays the 2D plottogether with the projections along the isotropic F₁ (t₁) andanisotropic F₂ (t₂) dimensions, respectively. In order to reduce thenoise in the projection along the isotropic dimension, only the part inthe 2D plot containing the spectral information was used, i.e. theinformation inside the band indicated in FIG. 9A. In this way thesignal-to-noise was enhanced by a factor of 3-4 compared with the casethat the full areas were used to generate the projection. By makingslices parallel to the F₂ axis, the anisotropic line shapes of eachisotropic peak can be determined separately, nine of which are plottedin FIG. 9B.

It follows from FIGS. 9A and 9B that substantial line narrowing isobtained with PHORMAT. For example, the width of the anisotropic line ofthe methyl peak at 0.9 ppm in FIG. 9B is about 150 Hz, while theisotropic line width is about 15 Hz, indicating that a line narrowingfactor of about 10 has been achieved.

FIGS. 10A, 10B, 10C and 10D show spectra of the fresh liver sampleobtained with different methods. FIG. 10A shows the anisotropic (F₂)projection of the PHORMAT 2D spectrum (cf. FIG. 9A), which is the sameas the spectrum obtained on a static sample. FIG. 10B shows theisotropic projection (F₁) projection of the PHORMAT 2D spectrum, givenin FIG. 9A. FIG. 10C displays the centerband spectrum obtained by a2D-PASS sequence that included a DANTE sequence and was acquired at aspinning rate of 40 Hz. Sixteen evolution steps were acquired, each ofwhich has 32 accumulations with a recycle time of 1.4 s. FIG. 10D showsa spectrum obtained with a comparative standard fast MAS at a spinningrate of 4 kHz with water suppression using DANTE, which was acquiredusing the PASS sequence with the five 180° pulses equally spaced apartduring a period of 25 ms. Thirty-two scans with a recycle delay time of1.4 s were used.

It follows that significant resolution enhancements were obtained byPHORMAT, PASS, and fast MAS. However, the 2D-PASS at a spinning rate of40 Hz (FIG. 10C) gives the best resolution, even better than that offast MAS (FIG. 10D), where additional B₀ inhomogeneity broadening mayhave been induced by the spinning itself. It is estimated that comparedwith the line widths observed with PASS, the widths in a MAS and PHORMATexperiment are increased by 2 and 5 Hz, respectively. This reducedspectral resolution in PHORMAT may be due to experimental imperfectionssuch as an error in the rotor markings, residual anisotropy in thesample packing, short-term spinning instability, and the drift of themain magnetic field (no field lock was applied during the relativelylong measuring time (3 hours)). Moreover, the increased broadening maybe caused by molecular diffusion during the evolution and storage timeof the magnetization.

FIGS. 11A, 11B, 11C and 11D show the ¹H spectra of the treated and agedliver sample obtained with 1 Hz PHORMAT (FIGS. 11A, 11B), 40 Hz 2D-PASS(FIG. 11C), and 4 kHz MAS (FIG. 11D). The PHORMAT results were acquiredusing the same experimental parameters as those with respect to FIGS. 9Aand 9B except that the number of evolution increments was doubled to 200to accommodate the increased line narrowing in the aged sample. The2D-PASS results were acquired using the same experimental parameters asthose with respect to FIG. 10C except that the accumulation number foreach evolution increment is increased to 64. The MAS results wereacquired using the same experimental parameters as those with respect toFIG. 10D except that the number of scans was increased to 96.

In this sample all experiments produced spectra with a significantlyhigher resolution than in the fresh untreated sample. More than 23 peakscan be distinguished in FIG. 11B, four of which are highlighted in thefigure. These peaks correspond to 1): Choline methyl, 2): phosphocholinemethyl and 3&4): glucose and trimethylamine-N-oxide methyl. It followsfrom FIGS. 11A and 11B, which display the anisotropic (F₂) and isotropic(F₁) projections of the PHORMAT experiment, respectively, that also inthe aged sample substantial line narrowing is obtained with PHORMAT. Forexample, the average anisotropic line width of peaks 1-4 is about 55 Hz,while the isotropic line widths of these peaks is about 4 Hz. Hence inthis sample the narrowing factor is approximately 14, comparable to thefactor 10 obtained in the fresh sample. These isotropic widths approachthose observed with 2D-PASS (2 Hz) and fast MAS (3 Hz). Again 2D-PASS(FIG. 4c) offers the highest resolution, consistent with the resultsobtained on the fresh sample.

It follows from the above that substantial spectral resolutionenhancements can be obtained with PHORMAT. The above-detailed resultsare unexpectedly good, as it was anticipated that the diffusion of themolecules in the susceptibility gradients would result in severe linebroadening at lower spinning speeds.

Having illustrated and described the principles of our invention withreference to several preferred embodiments, it should be apparent tothose of ordinary skill in the art that the invention may be modified inarrangement and detail without departing from such principles.

What is claimed is:
 1. A method of performing a magnetic resonanceanalysis of a biological object comprising: placing the biologicalobject in a main magnetic field and in a radio frequency field, the mainmagnetic field having a static field direction; rotating the biologicalobject at a rotational frequency of less than about 100 Hz around anaxis positioned at an angle of about 54°44′ relative to the mainmagnetic static field direction; pulsing the radio frequency to providea pulse sequence that includes a phase-corrected magic angle turningpulse segment and a water suppression pulse segment; and collecting datagenerated by the pulsed radio frequency.
 2. A method according to claim1 wherein the water suppression pulse segment comprises a DANTEsequence.
 3. A method according to claim 1 wherein the biological objectis rotated at a rotational frequency of about 1 to about 100 Hz.
 4. Amethod according to claim 1 wherein the main magnetic field remainsstationary during the biological object rotation.
 5. A method accordingto claim 1 wherein the phase-corrected magic angle turning pulse segmentincludes at least a first 90° pulse, a second 90° pulse, and a third 90°pulse, and the water suppression pulse segment is performed immediatelyprior to the third 90° pulse.
 6. A method according to claim 1 whereinthe biological object is rotated at a rotational frequency of about 1 toabout 50 Hz.
 7. A method according to claim 1 wherein the biologicalobject comprises a fluid object.
 8. A method of performing a magneticresonance analysis of a biological object comprising: placing thebiological object in a main magnetic field and in a radio frequencyfield, the main magnetic field having a static field direction; rotatingthe biological object at a rotational frequency of less than about 100Hz around an axis positioned at an angle of about 54°44″ relative to themain magnetic static field direction; pulsing the radio frequency toprovide a pulse sequence that includes a phase-corrected magic angleturning pulse segment comprising an initial π/2 pulse; and collectingdata generated by the pulsed radio frequency.
 9. A method of performinga magnetic resonance analysis of a biological object comprising:subjecting the biological object to a main magnetic field and a pulsedradio frequency field, the main magnetic field having a static fielddirection; rotating the biological object at a rotational frequency ofless than about 100 Hz around an axis positioned at an angle of about54°44′ relative to the main magnetic static field direction; controllingthe pulsed radio frequency to provide a sequence that includes aphase-corrected magic angle turning pulse segment and a watersuppression pulse segment; and generating a magnetic resonance analysisof the response by nuclei in the biological object to the pulsed radiofrequency sequence.
 10. A method according to claim 9 wherein thebiological object is rotated at a rotational frequency of about 1 toabout 100 Hz.
 11. A method according to claim 9 wherein the watersuppression pulse segment comprises a DANTE sequence.
 12. A methodaccording to claim 9 wherein the phase-corrected magic angle turningpulse segment includes an initial π/2 pulse.
 13. A method according toclaim 9 wherein the phase-corrected magic angle turning pulse segmentincludes at least a first 90° pulse, a second 90° pulse, and a third 90°pulse, and the water suppression pulse segment is performed immediatelyprior to the third 90° pulse.
 14. A method according to claim 9 whereinthe biological object is rotated at a rotational frequency of about 1 toabout 50 Hz.
 15. A method of performing a magnetic resonance analysis ofa biological object comprising: placing the biological object in a mainmagnetic field and in a radio frequency field, the main magnetic fieldhaving a static field direction; rotating the biological object at arotational frequency of less than about 10 Hz around an axis positionedat an angle of about 54°44′ relative to the main magnetic static fielddirection; pulsing the radio frequency to provide a pulse sequence thatincludes a magic angle turning pulse segment; and collecting datagenerated by the pulsed radio frequency.
 16. A method according to claim15 wherein the biological object comprises a fluid object.
 17. A methodaccording to claim 15 wherein the magic angle turning pulse segmentcomprises a phase-corrected magic angle turning pulse segment.
 18. Amethod according to claim 15 wherein the pulse sequence furthercomprises a water suppression pulse segment.
 19. A method according toclaim 15 wherein the biological object is rotated at a rotationalfrequency of less than about 3 Hz.
 20. A method according to claim 18wherein the water suppression pulse segment comprises a DANTE sequence.21. A method according to claim 17 wherein the phase-corrected magicangle turning pulse segment includes at least a first 90° pulse, asecond 90° pulse, and a third 90° pulse, the method further comprisingperforming a water suppression pulse segment immediately prior to thethird 90° pulse.
 22. A method of performing a magnetic resonanceanalysis of a biological object comprising: subjecting the biologicalobject to a main magnetic field and a pulsed radio frequency field, themain magnetic field having a static field direction; rotating thebiological object at a rotational frequency of less than about 10 Hzaround an axis positioned at an angle of about 54°44′ relative to themain magnetic static field direction; controlling the pulsed radiofrequency to provide a sequence that includes a magic angle turningpulse segment; and generating a magnetic resonance analysis of theresponse by nuclei in the biological object to the pulsed radiofrequency sequence.
 23. A method according to claim 22 wherein the magicangle turning pulse segment comprises a phase-corrected magic angleturning pulse segment.
 24. A method according to claim 22 wherein thesequence further comprises a water suppression pulse segment.
 25. Amethod according to claim 22 wherein the biological object is rotated ata rotational frequency of less than about 3 Hz.
 26. A method accordingto claim 24 wherein the water suppression pulse segment comprises aDANTE sequence.
 27. A method according to claim 23 wherein thephase-corrected magic angle turning pulse segment includes at least afirst 90° pulse, a second 90° pulse, and a third 90° pulse, the methodfurther comprising performing a water suppression pulse segmentimmediately prior to the third 90° pulse.
 28. A method of performing amagnetic resonance analysis of a biological fluid object comprising:placing the biological fluid object in a main magnetic field and in aradio frequency field, the main magnetic field having a static fielddirection; rotating the biological fluid object at a rotationalfrequency of about 20 to about 100 Hz around an axis positioned at anangle of about 54°44′ relative to the main magnetic static fielddirection; pulsing the radio frequency to provide a pulse sequence thatincludes a 2D-phase-altered spinning sidebands pulse segment; andcollecting data generated by the pulsed radio frequency.
 29. A method ofperforming a magnetic resonance analysis of a biological fluid objectcomprising: placing the biological fluid object in a main magnetic fieldand in a radio frequency field, the main magnetic field having a staticfield direction; positioning the biological fluid object along a magicaxis located at an angle of about 54°44′ relative to the main magneticstatic field direction; reorienting the biological fluid object aboutthe magic angle axis between three predetermined positions, the threepredetermined positions being related to each other by 120°; pulsing theradio frequency to provide a pulse sequence capable of producing aspectrum that is substantially free of anisotropic broadening; andcollecting data generated by the pulsed radio frequency.
 30. A methodaccording to claim 29 wherein the magnetic resonance analysis comprisesmagnetic resonance imaging and the radio frequency pulse sequenceincludes a magic angle turning pulse segment, the method furthercomprising: placing the biological object in at least one pulsedmagnetic field gradient; pulsing the radio frequency and pulsed magneticfield gradient to generate spatially-selective nuclear magneticresonance data; and generating a magnetic resonance analysis of theresponse by nuclei in the biological object to the pulsed radiofrequency sequence.
 31. A method of performing a magnetic resonanceanalysis of a biological object comprising: providing a main magneticfield that includes a first component having a static field directionand an amplitude and a second and a third component, each second andthird component having a sinusoidal field in a plane perpendicular tothe static field direction of the first component and with an amplitudethat is 2^(½) times the amplitude of the static field of the firstcomponent, wherein the second and third components produce a magneticfield that rotates in a plane perpendicular to the static fielddirection at a frequency of less than about 100 Hz resulting in anoverall field that is rotating around an axis located at an angle ofabout 54°44′ relative to the static field direction of the firstcomponent; placing the biological object in the main magnetic field andin a radio frequency field; pulsing the radio frequency to provide apulse sequence capable of producing a spectrum that is substantiallyfree of spinning sideband peaks; and collecting data generated by thepulsed radio frequency.
 32. A method according to claim 31 wherein themagnetic resonance analysis comprises magnetic resonance imaging and theradio frequency pulse sequence includes a magic angle turning pulsesegment, the method further comprising: placing the biological object inat least one pulsed magnetic field gradient; pulsing the radio frequencyand pulsed magnetic field gradient to generate spatially-selectivenuclear magnetic resonance data; and generating a magnetic resonanceanalysis of the response by nuclei in the biological object to thepulsed radio frequency sequence.
 33. A method according to claim 31wherein the biological object comprises a fluid object.
 34. A method ofperforming a magnetic resonance analysis of a biological objectcomprising: placing the biological object in a main magnetic field andin a radio frequency field, the main magnetic field having a staticfield direction; mechanically rotating a magnet around an axis at anangle of about 54°44′ relative to the main magnetic static fielddirection at a rotational frequency of less than about 100 Hz; pulsingthe radio frequency to provide a pulse sequence capable of producing aspectrum that is substantially free of spinning sideband peaks; andcollecting data generated by the pulsed radio frequency.
 35. A methodaccording to claim 34 wherein the magnetic resonance analysis comprisesmagnetic resonance imaging and the radio frequency pulse sequenceincludes a magic angle turning pulse segment, the method furthercomprising: placing the biological object in at least one pulsedmagnetic field gradient; pulsing the radio frequency and pulsed magneticfield gradient to generate spatially-selective nuclear magneticresonance data; and generating a magnetic resonance analysis of theresponse by nuclei in the biological object to the pulsed radiofrequency sequence.
 36. A method according to claim 34 wherein thebiological object comprises a fluid object.
 37. A method of performing amagnetic resonance analysis of a biological object comprising: placingthe biological object in a main magnetic field and in a radio frequencyfield, the main magnetic field having a static field direction; rotatingthe biological object at a rotational frequency of less than about 50 Hzaround an axis positioned at a magic angle of about 54°44′ relative tothe main magnetic static field direction; rotating the main magneticfield at a rotational frequency of less than about 50 Hz around themagic angle axis such that the main magnetic field and the biologicalobject rotate simultaneously in an opposite rotational direction;pulsing the radio frequency to provide a pulse sequence capable ofproducing a spectrum that is substantially free of spinning sidebandpeaks; and collecting data generated by the pulsed radio frequency. 38.A method according to claim 37 wherein the magnetic resonance analysiscomprises magnetic resonance imaging and the radio frequency pulsesequence includes a magic angle turning pulse segment, the methodfurther comprising: placing the biological object in at least one pulsedmagnetic field gradient; pulsing the radio frequency and pulsed magneticfield gradient to generate spatially-selective nuclear magneticresonance data; and generating a magnetic resonance analysis of theresponse by nuclei in the biological object to the pulsed radiofrequency sequence.
 39. A method according to claim 37 wherein thebiological object comprises a fluid object.
 40. A method of magneticresonance imaging of a biological fluid object comprising: subjectingthe biological fluid object to a main magnetic field, a pulsed radiofrequency field, and at least one pulsed magnetic field gradient, themain magnetic field having a static field direction; rotating thebiological fluid object at a rotational frequency of less than about 100Hz around an axis positioned at an angle of about 54°44″ relative to themain magnetic static field direction; controlling the pulsed radiofrequency to provide a pulse sequence that includes a magic angleturning pulse segment; pulsing the pulsed radio frequency and pulsedmagnetic field gradient to generate spatially-selective nuclear magneticresonance data; and generating a magnetic resonance analysis of theresponse by nuclei in the biological fluid object to the pulsed radiofrequency sequence.
 41. A method according to claim 40 wherein thebiological fluid object is rotated at a rotational frequency of about 1to about 50 Hz, and the pulse sequence includes a phase-corrected magicangle turning pulse segment.
 42. A method according to claim 41 whereinthe biological fluid object is rotated at a rotational frequency of lessthan about 10 Hz.
 43. A method according to claim 40 wherein thebiological fluid object is rotated at a rotational frequency of at leastabout 20 Hz, and the pulse segment includes a 2D-phase-altered spinningsidebands pulse segment.
 44. A method of magnetic resonance imaging of abiological fluid object comprising: placing the biological fluid objectin a main magnetic field and in a radio frequency field, the mainmagnetic field having a static field direction; rotating the biologicalfluid object at a rotational frequency of less than about 100 Hz aroundan axis positioned at an angle of about 54°44′ relative to the mainmagnetic static field direction; pulsing the radio frequency to providea pulse sequence that includes a phase-corrected magic angle turningpulse segment; and collecting data generated by the pulsed radiofrequency.
 45. A method according to claim 44 wherein the pulse sequencefurther comprises a water suppression pulse segment.
 46. A methodaccording to claim 44 wherein the biological fluid object is rotated ata rotational frequency of about 1 to about 50 Hz.
 47. A method ofmagnetic resonance imaging of a biological object comprising: subjectingthe biological object to a main magnetic field, a pulsed radio frequencyfield, and at least one pulsed magnetic field gradient, the mainmagnetic field having a static field direction; rotating the biologicalobject at a rotational frequency of less than about 10 Hz around an axispositioned at an angle of about 54°44′ relative to the main magneticstatic field direction; controlling the pulsed radio frequency toprovide a pulse sequence that includes a magic angle turning pulsesegment; pulsing the pulsed radio frequency and pulsed magnetic fieldgradient to generate spatially-selective nuclear magnetic resonancedata; and generating a magnetic resonance analysis of the response bynuclei in the biological object to the pulsed radio frequency sequence.48. A method according to claim 47 wherein the pulse sequence furthercomprises a water suppression pulse segment.
 49. A method according toclaim 47 wherein the water suppression pulse segment comprises a DANTEsequence.
 50. A method of magnetic resonance imaging of a biologicalobject comprising: subjecting the biological object to a main magneticfield, a pulsed radio frequency field, and at least one pulsed magneticfield gradient, the main magnetic field having a static field direction;rotating the biological object at a rotational frequency of less thanabout 100 Hz around an axis positioned at an angle of about 54°44′relative to the main magnetic static field direction; controlling thepulsed radio frequency to provide a pulse sequence that includes a magicangle turning pulse segment and a water suppression pulse segment;pulsing the pulsed radio frequency and pulsed magnetic field gradient togenerate spatially-selective nuclear magnetic resonance data; andgenerating a magnetic resonance analysis of the response by nuclei inthe biological object to the pulsed radio frequency sequence.
 51. Amethod according to claim 50 wherein the water suppression pulse segmentcomprises a DANTE sequence.
 52. A method according to claim 50 whereinthe biological object is rotated at a rotational frequency of about 1 toabout 50 Hz, and the pulse sequence includes a phase-corrected magicangle turning pulse segment.
 53. A method according to claim 50 whereinthe biological object is rotated at a rotational frequency of at leastabout 20 Hz, and the pulse segment includes a 2D-phase-altered spinningsidebands pulse segment.